Aspergillus fumigatus spores (AFsp) is associated with an inflammatory response, potentially leading to allergic and/or chronic pulmonary aspergillosis. The aim of our study is to better understand the host response, first in vitro, then in vivo, following the chronic exposure of mice to AFsp. We investigated the inflammatory response to AFsp in cell mono- and co-culture systems with murine macrophages and alveolar epithelial cells. The mice were subjected to two intranasal instillations using 105 AFsp. Their lungs were processed for inflammatory and histopathological analyses. In cell culture, the gene expressions significantly increased for TNF-α, CXCL-1, CXCL-2, IL-1β, IL-1α and GM-CSF in macrophages, with these increases being limited for TNF-α, CXCL-1 and IL-1α in epithelial cells. In co-culture, increases in the TNF-α, CXCL-2 and CXCL-1 gene expressions were observed to be associated with increased protein levels. The in vivo lung histological analyses of mice challenged by AFsp showed cellular infiltrates in the peribronchial and/or alveolar spaces. A Bio-Plex approach on the bronchoalveolar lavage revealed significant increases in the protein secretion of selected mediators of the challenged mice compared to the unchallenged mice. In conclusion, the exposure to AFsp resulted in a marked inflammatory response of macrophages and epithelial cells. These inflammatory findings were confirmed in mouse models associated with lung histologic changes.The airway exposure to  Aspergillus fumigatus is a filamentous airborne saprophytic and ubiquitous fungus. The exposure to its spores was associated with lung airway inflammation in subjects living in damp dwellings or exposed to professional/industrial environments [Aspergillus complications, which worsens their symptoms and contributes to increased morbidity and mortality [A. fumigatus spores such as neutropenic patients [ronments ,2, leadironments ,4. Patieortality ,6. The wortality . Chronicpatients . Of relepatients ,10. Furtpatients ,12.Aspergillus fumigatus spores are usually eliminated via mucociliary clearance and resident macrophages, which are actors of the innate immune mechanisms [Aspergillus fumigatus and the underlying mechanisms of ABPA or CPA development in the immunocompetent host are not completely understood.In some patients, especially those with asthma, a link between a sensitization to fungi and the severity of the disease and frequency of exacerbations was described, contributing to poor clinical outcomes. In immunocompetent patients, daily inhaled chanisms ,13,14,15chanisms . Both thA. fumigatus spores. To this end, we first set up models of cell mono- and co-cultures using relevant lung cells, namely, alveolar epithelial cells and macrophages, that were exposed to A. fumigatus spores (AFsp), and we characterized their inflammatory responses to AFsp. Then, we developed a mouse model of the sensitization to AFsp, with week 1 and week 6 intranasal instillations of the spores, to further investigate the changes in the host lung inflammation and tissue.Thus, the aim of the present study is to improve our understanding of the lung response to Our study highlighted a strong inflammatory response in the monocultures of RAW 264.7 macrophages and the MLE-15 epithelial cells, and in the co-cultures of both cells. Interestingly, the cell response was different for the cells that were in monocultures compared with the cells that were in co-cultures, with a reduction in the expression of IL-1α in the co-culture compared to RAW 264.7 alone, and in contrary, a synergistic effect was observed for TNF-α, which was more increased in the co-cultured cells versus the monocultured cells. For the in vivo model, the mice elicited a robust inflammatory response, which was correlated with the pathological changes in the lung tissue.® TIB-71™, CLS Cat# 400319/p462_RAW-2647, RRID:CVCL_0493) and MLE-15 lung epithelial cells  were cultured in DMEM culture media  supplemented with 10% fetal bovine serum  and 1% Penicillin/Streptomycin (Life Technologies) at 37 °C in a humidified atmosphere containing 5% CO2. Cells were washed with sterile phosphate-buffered saline (PBS) (Life Technologies), then treated at sub-confluence (80%) with A. fumigatus spores at a 1:3 ratio (cell/spores). A. fumigatus CBS144.89 strain spores were prepared as previously described [®, St. Louis, MO, USA). The suspension was counted by using a Kova® hemacytometer and calibrated to achieve the 1:3 ratio (cells:spores). A Pseudomonas aeruginosa lipopolysaccharide (LPS) at 1 μg/mL served as a positive control. Four hours post-treatment, RNA extraction and RT-qPCR were performed on cells. For each condition, cells were stained with Kwik-Diff Stain kit for cytologic analysis . Three independent experiments for both monocultures were performed.Cell monocultures: Murine RAW 264.7 macrophages , after epithelial cell count. On treatment day, the cells were subjected to similar treatment with In parallel experiments, for co-culture control, cells were fixed with methanol and immunostained using the specific antibodies anti-CD68  for the macrophages and anti-E-cadherin  for epithelial cells. For secondary antibodies, Alexa Fluor™ 594 and 488 (Thermo Fisher) were used, respectively. Four independent experiments were performed.Aspergillus fumigatus spores (AFsp group), both at week 1 and week 6. Mice were sacrificed 24 h after the last instillation, and their blood (serum), bronchoalveolar lavage (BAL) and lungs were recovered as previously described to perform inflammatory (left lobe) and histopathological analyses (right lobes) [Animal experiments: A total of 14 C57BL/6J Specific and Opportunistic Pathogen Free (SOPF) mice  were purchased from Charles River Laboratories. Mice were housed in ventilated racks for animal facilities with food and water ad libitum and a 12 h dark/light cycle. After a week of acclimatization period, mice were randomly divided into the following two groups with equal sex ratio: a control group exposed to two intranasal instillations of phosphate-buffered saline (PBS) (control group), and a spore group exposed to two intranasal instillations of t lobes) .Ethical considerations: All animal experiments complied with the European Union Directive 2010/63/EU on the protection of animals used for scientific purposes. The protocol was approved by the French Ministry of Higher Education and Research after agreement from the local Ethical Committee for experiments on animals .Aspergillus fumigatus spore preparation and intranasal instillation: A. fumigatus CBS144.89 strain spores were prepared as described for cell culture experiments [® hemacytometer and calibrated to a final concentration of 105 conidia/50 µL in PBS + 0.01% Tween 20. The spore suspension obtained was administered via intranasal instillation in the two nares of the mice, after mice were anesthetized with isoflurane at 3%.eriments . The susAspergillus DNA detection: DNA extraction from lung was performed using an automaton Maxwell®16  after grinding by using Roche MagNA Lyser  beads and eluted in 100 µL. DNA extracts were tested in duplicate with an A. fumigatus-specific qPCR as previously reported in [A. fumigatus spores were used, and a standard curve was established between the threshold cycle (Ct) values and the spore numbers; this standard curve was used to estimate the spore number via interpolation of the Ct value obtained using real-time PCR [orted in  by usingorted in . For quatime PCR .Histopathological analysis: Pulmonary samples (right lobes) were processed for all 14 mice included. All samples were fixed in formalin , split in four sections and embedded in paraffin . For each sample, two 3 µm thick tissue sections of the paraffin block were performed. A histopathological analysis was performed on both tissue sections by a pathologist (A.T) and an experienced researcher (A.Be.), blind to the randomization of mice, after staining with Hematoxylin–Eosin–Saffron (HES) stain  for tissue analysis, and Grocott stain for fungal morphology analysis  according to the manufacturer’s recommendations. The microscopic analysis included the description of the following: presence/absence of tissue inflammation, location of the inflammation, type of inflammatory infiltrate  and semi quantitative inflammation quantification , presence/absence of perivascular edema and presence/absence of fungal elements on the slide stained via Grocott. Two independent readers read all slides.All histopathological photographs for original figures of the present article were taken after slide scanning using Aperio AT2 scanner  and Image Scope software (Leica Biosystems).®) and Nucleospin RNA set for NucleoZOL  according to the manufacturer’s protocol. DNase free kit (Thermo Fisher) was used to eliminate residual gDNA. cDNA was obtained via reverse transcription using High-Capacity cDNA Reverse transcription kit (Thermo Fisher). TaqMan® real-time quantitative polymerase chain reaction (RTqPCR) assays were performed using the corresponding cDNAs on an AriaMx Real-Time PCR thermocycler . The hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene was used as endogenous control , and Quantikine® CXCL-1 and CXCL-2  were performed using supernatants collected from co-culture cell experiments or BAL samples (CXCL-2 and IgE) from mice according to the manufacturer’s protocol.Enzyme-linked immunosorbent assay (ELISA): ELISA MAX™ Deluxe Set on TNF-α, IL-1α and IgE . The assays were performed by following the manufacturer’s protocol. The BAL were not diluted, and sera were diluted to a quarter.Cytotoxicity: Cytotoxicity assay was performed using CyQUANT™ LDH Cytotoxicity Assay kit (Thermo Fisher). Assays were performed on supernatants of co-cultured cells and on BAL from spore and control groups following the manufacturer’s protocol.Statistics: All statistical analyses were performed using Prism version 8.4.2 software , and data were represented as mean with standard deviation (SD) of minimum three independent experiments.p < 0.05 was considered statistically significant.Statistical analyses were made on ΔCt for RT-qPCR and concentrations (pg/mL) for ELISA and BioPlex assays. To compare the difference between the 2 groups, normality was verified using Shapiro–Wilk test; then, either Student’s unpaired t-test or Welch’s test or Mann–Whitney test were performed. A To assess the inflammatory response of the first sentinel cells involved in the immune reaction, we evaluated the mRNA expression profile of the different cytokines in the AFsp-exposed RAW 264.7 macrophages and the MLE-15 epithelial cells. The LPS-treated cells were used as positive controls .p = 0.0179), TNF-α , CXCL-2 , CXCL-1 , GM-CSF  and IL-1α  after the exposure to A. fumigatus spores , CXCL-1  and IL-1α  after the exposure to A. fumigatus spores (p = 0.0898) was observed.In the MLE-15 cells, significant gene expression increases were observed for TNF-α , 17-fold for TNF-α (p = 0.0005), 233-fold for CXCL-2 (p = 0.0003), 31-fold for CXCL-1 (p = 0.0039), 9-fold for GM-CSF (p = 0.0110) and 55-fold for IL-1α (p = 0.0004)  B.p < 0.0001), CXCL-2  and CXCL-1  (p = 0.7326) in the percentage of cellular viability between the controls and the cells exposed to the spores  C. Howevee spores .Aspergillus invasive infection.After the acute exposure of macrophages and epithelial cells to AFsp, we aimed to study the inflammatory response of sensitized mice to AFsp after two instillations 5 weeks apart, mimicking repeated exposures with a long follow-up of these immunocompetent mice that are not susceptible to Aspergillus fumigatus spores. No statistical difference was observed in the sex-dependent comparison either  cases A–E. The In the control group, inflammation was observed in only 1/7 (14.3%) cases. The inflammatory reaction was composed of lymphocytes and plasma cells and was perivascular and peribronchiolar. No perivascular edema were observed G–I. All p = 0.0399) compared to the control group. Macrophages represented the dominant cell type and was significantly increased in the AFsp group compared to the control group (p = 0.0046).To explore the underlying basis of the inflammatory reaction in tissue, we investigated the inflammatory cell recruitment in the mice exposed to AFsp. The total cell counts in the BAL A of the A. fumigatus spores was evidenced by A. fumigatus-specific qPCR in the lungs of the mice exposed to the spores, whereas no fungal DNA was observed in the control group. The mean fungal burden in the mice challenged with A. fumigatus spores was 428.8 spore-equivalents/mL , IL-1α , CXCL-1 , GM-CSF , IL-1β , IL-6 , MIP-1α , and MIP-1β . Although not statistically significant, an increasing trend in the TNF-α gene expression  was observed.To corroborate the results obtained in vitro, we tested the levels of gene expression of the same mediators in the lungs. In the mice exposed to videnced  for CXCLTo further explore the inflammatory response, we assessed the levels of secretion of several mediators in the BAL of the AFsp mice using a multiplex immunoassay approach.p = 0.3) and ELISA showed an absence of IgE in the BAL .Significant increases in the secretion of 17 proteins were found in the BAL  from thep = 0.0490), IL-5 , IL-9 , IL-13 , GM-CSF , CXCL-1 , RANTES (=CCL5)  and Eotaxin .To assess the systemic inflammation, we tested the sera from the AFsp and control mice groups using the same multiplex immunoassay. A significant increase in the secretion of several proteins was observed in the AFsp mice  as folloA. fumigatus spores, we developed the following models: monocultures then co-cultures with macrophages and epithelial cells, relevant lung cells involved in immune response and an animal model. The AFsp exposure was performed in the culture model while the sensitization to AFsp was mimicked in the mouse model with two AFsp instillations 5 weeks apart, with a long mouse follow-up (6 weeks).To better understand the pulmonary and systemic inflammatory response of immunocompetent hosts to We assessed the inflammatory response of the murine macrophages and alveolar epithelial cells, individually or co-cultured, to AFsp. Our rationale was to closely mimic the cross-talk, if any, between these two cell types that are known to act as sentinels for the immune system in the lungs.IL-1α, IL-1β, CXCL-2, TNF-α, CXCL-1 and GM-CSF) via murine macrophages, which are key cells for the innate immune response. A weaker induction of proinflammatory cytokine gene expression  was observed for the epithelial cells, compared to the macrophages, following exposure to AFsp. All of these mediators are involved in immune cell recruitments, especially neutrophils [A. fumigatus from the airway [7 environmental A. fumigatus spores led to a significant increase in the TNF-α gene expression, with the maximal at 6 h with a two-fold induction, and no change in the IL-1β gene expression regardless of the duration of treatment and the concentrations of spores [A. fumigatus spores, and only 4 h was sufficient to start up an immune response in each monoculture. Additionally, we wanted to investigate the inflammatory response of the cells in the co-culture.Our results highlighted, as expected, a strong induction of proinflammatory cytokine gene expression  in the broncho-alveolar lavage either. It might not be secreted in this condition, or it might serve for other genes or the regulation of other proteins.Our study evidenced a marked inflammatory response to the Aspergillus fumigatus focused on one cell type in particular, either epithelial cells, macrophages or fibroblasts, but rarely on a more complex model, where several cell types are in contact to try to recreate the pulmonary environment. Our co-culture model makes it possible to study the inflammatory response after the exposure to fungal spores by approaching the in vivo model with the two cell types involved in the first line of defense against pathogens.Most studies on A. fumigatus spores in immunocompetent hosts, mice were challenged with two exposures of 105 spores 5 weeks apart. Most studies with the exposure of mice to A. fumigatus spores focused on an invasive pulmonary aspergillosis model in immunocompromised mice [A. fumigatus before using spores [A. fumigatus allergens [To better understand the pulmonary inflammatory response to sed mice , or on ag spores , or withllergens ,33, or ullergens  or intrallergens . To our Indeed, in histological analyses, inflammation was reported for 6/7 cases and composed of lymphocytes, plasma cells and some neutrophils/eosinophils, and perivascular edema was present for all of the mice exposed to the spores.IL-1β, CXCL-2, CXCL-1, GM-CSF, IL-6, MIP-1α and MIP-1β, and correlated with their up-secretion in the BAL.The inflammation was also supported in the lung by the expression of several genes such as A. fumigatus spores [A. fumigatus spores [A. fumigatus spores 5 days apart are not sufficient to induce a pulmonary allergic response without a sensitizing event, but it was performed with a different strain of mice (BALB/c), with a different strain of spores (ATCC 13073) and with two-log more spores than in our experiment [A. fumigatus-induced cytokine response with similarities to an allergic airway disease. However, most studies showed an increase in the IgE in the sera of mice exposed to Aspergillus fumigatus. In our study, we did not find these results in the BAL, but this might be due to the fact that the IgE half-life in adult mice is only 12 h [Aspergillus fumigatus spores in immunocompetent hosts to develop new therapeutic solutions against the potentially deleterious immune response. The mechanism of inflammation is complex and involves both immune cells and epithelial cells, which are not only useful for mucociliary clearance, but also play a major role in inflammation.Interestingly, in our in vivo study, the other mediators screened in the multiplex immunoassay were increased as follows: IL-4, a cytokine secreted particularly by eosinophils and which plays a role in the regulation of antibody production, is known to enhance the secretion and cell surface expression of IgE and IgG1 ,37, and s spores ; IL-5, as spores ; IL-13, s spores , stimulas spores ; eotaxins spores ; MCP-1 (s spores ; MIP-1α s spores ; MIP-1β s spores  and RANTs spores . All of s spores . These mperiment . Mice caperiment . In our nly 12 h . It is i


Therapeutic options for breast cancer patients with brain metastases (BM)/leptomeningeal carcinomatosis (LMC) are limited. Here, we report on the effectiveness and safety of trastuzumab deruxtecan (T-DXd) in human epidermal growth factor receptor 2-positive breast cancer patients with BM. Data were analyzed for 104 patients administered T-DXd. Overall response rate (ORR), progression-free survival (PFS), overall survival (OS), intracranial (IC)-ORR, and IC-PFS were evaluated. ORR by investigator assessment was 55.7% . Median PFS was 16.1 months; 12-month OS rate was 74.9% . Median time-to-treatment failure was 9.7 months. In 51 patients with BM imaging, IC-ORR and median IC-PFS by independent central review were 62.7% and 16.1 months, respectively. In 19 LMC patients, 12-month PFS and OS rates were 60.7% and 87.1%, respectively. T-DXd showed effectiveness regarding IC-ORR, IC-PFS, PFS, and OS in breast cancer patients with BM/active BM, and sustained systemic and central nervous system disease control in LMC patients.Trial Registration: UMIN000044995. For BM specifically, the incidence has been reported to be as high as 31% in patients with HER2-positive breast cancer2. Breast cancer patients with BM and/or LMC have limited treatment options.Breast cancer is one of the most common causes of central nervous system (CNS) metastases, including brain metastases (BM) and leptomeningeal carcinomatosis (LMC), and patients with CNS metastasis have a poor prognosis. The molecular subtype of breast cancer has been shown to have an impact on the incidence of CNS metastases. For example, the incidence of CNS metastasis was found to be twice as high in patients with human epidermal growth factor receptor 2 (HER2)-positive breast cancer compared with HER2-negative breast cancer 6. The European Association of Neuro-Oncology–European Society for Medical Oncology guidelines recommend systemic therapy based on the primary tumor for most breast cancer patients, but there is a lack of evidence to support this7.Guidelines for breast cancer treatment recommend local therapy for patients with symptomatic BM, such as surgical resection or irradiation, and to continue with the same anti-HER2 therapy if the systemic clinical benefit persists9. In the DESTINY-Breast01 study (NCT03248492), trastuzumab deruxtecan (T-DXd) showed clinical activity in breast cancer patients with BM who had received ≥2 prior anti-HER2-based regimens or who were previously treated with trastuzumab emtansine (T-DM1)11. The DESTINY-Breast03 trial showed higher progression-free survival (PFS) and overall response rate (ORR) in HER2-positive breast cancer patients with BM who were treated with T-DXd compared with those treated with T-DM113. The results of the TUXEDO-1 and DEBBRAH trials also support the use of T-DXd in HER2-positive breast cancer patients with BM15.Based on the results of the HER2CLIMB trial (NCT02614794), combination therapy with tucatinib plus trastuzumab plus capecitabine was approved by the Food and Drug Administration (FDA) for the treatment of adult patients with unresectable, advanced, or metastatic HER2-positive breast cancer, including patients with BM, who have received at least one regimen of anti-HER2 therapy for metastatic breast cancern = 24 and n = 43, respectively), and sample sizes of patients with active BM in the TUXEDO-1 and DEBBRAH trials  were small compared with the HER2CLIMB trial (174 patients with active BM). There is currently a lack of solid evidence on the activity of T-DXd in CNS metastases, and real-world data for T-DXd would be particularly valuable due to weak prospective evidence.Of note, only patients with stable BM were included in the DESTINY-Breast01 and 03 studies  who were treated with T-DXd were enrolled in this study , 73 (70.2%) patients had active BM without LMC, 17 (16.3%) had active BM with LMC, 6 (5.8%) had stable BM, 2 (1.9%) had only LMC, and 6 (5.8%) were not classified . In total, 41 (39.4%) patients were still under treatment.The background patient characteristics are summarized in Table N = 104), the median PFS was 16.1 months   Fig. , and the -) Fig. . The OS The median time-to-treatment failure (TTF) was 9.7 months  Fig. . Discontn = 97), 51.7% (CR: 6.9%) in symptomatic BM patients (n = 29), and 57.4% (CR: 4.4%) in asymptomatic BM patients (n = 68) (Supplementary Table The ORR based on investigator assessment was 55.7% (complete response [CR]: 5.2%) in the total population (n = 51), the IC-ORR was 62.7%  evaluation by independent central review (ICR). The best overall response based on ICR assessment is summarized in Table N = 104)  at 12 months, and the median time-to-deterioration of CNS metastasis-related symptoms was not reached (- – -) in the total population . The IC-ORR (n = 51) was 62.7% , and the median IC-PFS (n = 89) was 16.1 months  in the patients with evaluable IC imaging  scoreThe current data set included 19 patients with LMC , with 12-month PFS and OS rates of 60.7%  and 87.1% , respectively. Nine patients with imaging data of the brain lesion were included in the IC evaluation by ICR. In patients with BM and LMC, the IC-ORR was 77.8% (7/9 patients).This was a study of 104 HER2-positive breast cancer patients with BM who were treated with T-DXd in a real-world clinical setting. Our findings suggest that T-DXd may be a treatment option for HER2-positive breast cancer patients with BM, including patients with LMC and active and stable BM, who are characterized by poor performance status.19. In the phase 2 LANDSCAPE trial, lapatinib and capecitabine for asymptomatic and oligosymptomatic patients with BM showed a response rate of 66% and time to whole brain radiotherapy of 8.3 months20. Tucatinib, trastuzumab, and capecitabine combination showed a response rate of 47.3% compared with 20% in the trastuzumab and capecitabine group among patients with active BM in the HER2CLlMB study9. Median PFS was improved from 4.1 months to 9.5 months, and OS from 11.6 months to 20.7 months with the addition of tucatinib. Based on this study, the combination of tucatinib, trastuzumab, and capecitabine is now regarded as the preferred systemic treatment option for active BM in patients with HER2-positive breast cancer21.In previous studies of the tyrosine kinase inhibitors lapatinib and neratinib in HER2-positive breast cancer patients with BM, PFS was less than 6 months and severe disease was not well controlled22. Clinical data on the potential activity of antibody–drug conjugates in stable and active BM were reported. The response rate with single-agent T-DM1 was 49.3% in the KAMILLA study, including the subset of patients with measurable BM without previous local radiotherapy23. In the DESTINY-Breast03 trial, which included 43 patients with stable BM, the PFS was 15 months, and the response rate was 80%12. The TUXEDO-1 trial was a single-arm prospective study of patients with newly diagnosed or progressive active BM treated with T-DXd. The response rate was 73.3% (11/15), median PFS was 14 months, and median OS was not reached at a median follow-up of 12 months14. The median PFS in the present study (16.1 months) was comparable to that in the BM subgroup of the DESTINY-Breast03 study (15 months) and numerically longer than that in the BM subgroup of the HER2CLIMB trial (9.5 months). Although the proportion of patients with BM was small, the IC-ORR was 62.7% in patients with measurable BM. The IC-CBR was 70.6% in the present study, suggesting that clinical benefit could be obtained in most patients. The median IC-PFS was higher (16.1 months) compared with that reported in the HER2CLIMB trial (9.9 months)9. In a recent retrospective multicenter cohort study of 17 patients with mainly HER2-positive metastatic breast cancer with BM24, the CNS-ORR and the 12-month CNS-PFS were reported to be 73% (11/15) and 74.7%, respectively, which is slightly higher than the IC-ORR and 12-month IC-PFS in the present study .Despite assumptions that monoclonal antibodies do not cross the blood–brain barrier, a radioisotope form of trastuzumab, imaging [64Cu] DOTA-trastuzumab, has been shown to localize in the BM of patients with HER2-positive metastatic breast cancer. In the PATRICIA study, pertuzumab plus high-dose trastuzumab (6 mg/kg weekly) in patients with progressive BM showed clinical benefit. The CNS ORR was 11% with four partial responses . The CBR at 4 and 6 months were 68% and 51%, respectively17. Our study showed no differences in PFS and OS between GPA score subgroups, meaning that T-DXd showed consistent effectiveness in terms of PFS and ORR in breast cancer patients with poor prognosis. The effectiveness of T-DXd for the treatment of IC lesions of breast cancer patients with active BM was similar to that in patients with stable BM, and T-DXd may provide some clinical benefit in patients with both active and stable BM.Of note, our study included patients with poor Karnofsky performance status scores who are usually excluded from prospective trials, such as those with GPA scores of 1.5–2.025. In a previous phase 2 single-arm study that evaluated the efficacy of the combination of tucatinib plus trastuzumab and capecitabine in patients with HER2-positive breast cancer and newly diagnosed LMC, the median time to CNS progression was 6.9 months, and the median OS time was 11.9 months26. In the present study, both the median PFS and median OS in patients with LMC were not reached. PFS and OS at 12 months were 60.7% and 87.1%, respectively, in patients with LMC. These findings suggest that T-DXd was effective in patients with LMC, who were not responsive to local therapy and in whom pharmacotherapy was not expected to be effective. A recent retrospective study of eight evaluable patients with HER2-positive metastatic breast cancer and LMC showed a durable response with T-DXd27; 50% (4/8) of patients achieved partial response, 50% (4/8) had stable disease, and the median OS was 10.4 months. The response was centrally reviewed using the EORTC/RANO revised LM scorecard28. Of note, the DEBBRAH trial is still ongoing and is recruiting a small cohort of patients with LMC, which will provide prospective data on this rare subgroup15.Our data suggest a longer PFS in patients with LMC compared with that reported in previous studies. An analysis of the Epidemiological Strategy and Medical Economics MBC database showed a median OS of 5.6 months in patients with HER2-positive LMC10. This difference in the incidence of ILD is consistent with the results of an integrated analysis of DESTINY-Breast01 and DS8201-A-J101 (unpublished data) in which the incidence of ILD in Japanese patients was higher than that in the overall population. This is also supported by a pooled analysis of nine phase 1 and 2 T-DXd monotherapy studies in which enrollment in Japan was a significant risk factor for ILD29. No novel AEs leading to treatment discontinuation were observed in breast cancer patients with BM who were treated with T-DXd.In this study, the most common AE leading to treatment discontinuation was ILD (18.3%), and this incidence of ILD was slightly higher than that reported in the DESTINY-Breast01 study (13.6%)The present study has some limitations. This was not a prospective study. Because the presence or absence of BM was determined by the investigator, the possibility of reporting bias cannot be ruled out as to whether all BM patients were reported. LMC was diagnosed on imaging by ICR, and the presence of tumor cells in spinal fluid was not confirmed. The frequency of imaging evaluation was not specified, so the ORR and PFS may have been overestimated. For greater objectivity, the event onset in time-to-deterioration of CNS metastasis-related symptoms was defined as the date of treatment initiation and was not reported by physicians. Therefore, the impact of pharmacotherapy for symptom management may not have been properly assessed. In addition, as the response in patients with LMC was based on RECIST-based assessment of parenchymal BM lesion, the results regarding drug effectiveness should be interpreted with caution. Furthermore, the present study was conducted only in Japanese patients, which limits the generalizability of current findings to other ethnicities.In conclusion, T-DXd was effective in breast cancer patients with BM, regardless of the presence of active or stable BM or the presence of LMC, indicating that IC tumors may be controlled by T-DXd. T-DXd has a manageable AE profile and may have survival benefits in breast cancer patients with BM who have a poor prognosis (low Karnofsky performance status score). In a small number of breast cancer patients with LMC, sustained systemic disease control was achieved with T-DXd. The ongoing DESTINY-Breast12 study (NCT04739761) will confirm the efficacy of T-DXd for active BM in patients with HER2-positive breast cancer.®, Daiichi Sankyo Co., Ltd., Tokyo, Japan and AstraZeneca, Cambridge, UK) for the treatment of HER2-positive breast cancer especially in patients with BM as of December 31, 2020, of which 62 medical institutions had eligible patients for this study and agreed to participate in the study. Data from HER2-positive breast cancer patients with BM who received T-DXd treatment between May 25, 2020, and April 30, 2021, were collected from each participating institution using a medical record retrieval system. The principal investigators checked the medical records for all extracted patients, and using the continuous survey method, all the research patients who met all the inclusion criteria and did not fulfill the exclusion criteria were selected. The data cut-off date for survival and other information was October 31, 2021; data entry began on November 12, 2021, and information from medical records was entered retrospectively.This was a multicenter, retrospective, medical chart review study. To avoid selection bias, a questionnaire was sent to all 344 medical institutions in Japan regarding usage of T-DXd , as well as the individual ethics committees at each study center . Patients who expressed a desire not to participate in the study prior to data fixation and those who had received T-DXd from participation in a clinical trial were excluded.The following outcomes were evaluated for the total population: TTF, PFS, ORR based on investigator assessment, OS, and time-to-deterioration of CNS metastasis-related symptoms. The following outcomes were evaluated for the population with imaging data of the brain lesion: IC-ORR, IC-PFS, IC-DOR, and IC-CBR. Each of the outcomes is defined in Supplementary Table Brain imaging data  of brain lesions from study patients were subjected to ICR to determine the effectiveness of treatment with T-DXd. The ICR consisted of three independent radiologists (a chairperson and two members) who were skilled in Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 evaluation to maintain a certain standard of evaluation. Two ICR members evaluated half of the images each, and the chairperson made an overall judgment of the results. All brain images are collected centrally. For each study patient, comprehensive assessment data at the time of visit and the date of imaging at each time point (visits at which response or progression was confirmed/not confirmed) were determined by ICR. BM was classified into analytical active BM, analytical stable BM, and LMC by the ICR as follows. The detailed criteria are shown in Supplementary Table According to ICR, patients with increasing/growing tumors in two brain imaging comparisons before T-DXd administration were defined as “active by ICR”. In this study, those who had not undergone whole brain radiotherapy within 30 days before T-DXd administration and excluding LMC were defined as “analytical active BM”.The patients who were not classified as active and/or LMC by ICR were defined as “stable by ICR”. In this study, those who were defined as stable by ICR and radiated active BM were defined as “analytical stable BM”.Patients judged to have LMC by ICR were classified as “active with LMC” or “only LMC” and were defined as “LMC” in the analysis.The analysis populations in the present study included the total population, defined as the population who met all the inclusion criteria and did not meet the exclusion criteria , and the population with imaging data of the brain lesion, defined as the population who had multiple imaging data of the brain lesion which were evaluable for IC response assessed by ICR among the total population.Regarding background patient characteristics, frequencies and percentages were calculated for categorical variables and summary statistics were calculated for continuous data. For OS, TTF, IC-DOR, IC-PFS, and time-to-deterioration of CNS metastasis-related symptoms, the median duration and its 95% CIs were calculated using the Kaplan–Meier method. The point estimates of survival (6 and 12 months) and their 95% CIs were calculated. For ORR, IC-ORR, and IC-CBR, point estimates and their 95% CIs were calculated. Subgroup analyses were performed according to the presence or absence of concomitant BM symptoms and patients’ background characteristics, among others. All statistical analyses were performed using SAS software, version 9.4 .Supplementary Materialsnr-reporting-summary


The expansion of antiretroviral therapy (ART) eligibility could lead to earlier initiation of Human Immunodeficiency Virus (HIV) treatment and consequently reduce the risk of HIV-associated Kaposi Sarcoma (KS). We investigated the impact of changes in the Nigerian HIV treatment guidelines on KS incidence among adults enrolled in HIV care in Nigeria.We analyzed data of adults who enrolled for HIV care from January 2006 to December 2016 at one of Nigeria’s largest HIV treatment centers. Based on changes in HIV treatment guidelines, we classified 2006–2009 as the pre-expansion period and 2010–2016 as the post-expansion period. We used Kaplan Meier curves to compare the incidence of KS in the pre-expansion to the post-expansion period. We used Cox regression models to assess the hazard for incident KS between the two periods after adjusting for potential confounders.Among 14,479 patients with HIV, the overall KS incidence was 2.35; 95% CI 2.01–2.74/1,000 person-years. The incidence of KS decreased from 2.53 to 1.58 per 1,000 person-years from 2006 to 2009 to 2010–2016. In models adjusting for age, sex, CD4-T cell count, and ART use, the risk for KS remained lower in 2010–2016 compared to 2006–2009. In analyses restricted to time on ART, there was no significant difference in KS incidence between HIV patients who enrolled in 2006–2009 and 2010–2016 after adjusting for age, sex, and CD4 T-cell count.The expansion of ART eligibility was associated with a reduced incidence of HIV-associated KS among adults initiating HIV care in Jos, Nigeria. The reduction was likely driven by earlier enrollment for HIV care and ART initiation.The online version contains supplementary material available at 10.1186/s12885-023-11402-3. Antiretroviral therapy (ART) has significantly reduced the incidence of Kaposi sarcoma (KS) among people with Human Immunodeficiency Virus/ Acquired Immunodeficiency Syndrome (HIV/AIDS) globally –4. Yet, In response to evolving evidence showing significant survival benefits of earlier ART initiation –16, NigeIn this retrospective cohort study, we analyzed data of adults who initiated care at the Jos University Teaching Hospital (JUTH) HIV Clinic between January 1, 2006, and December 31, 2016, with follow-up until December 31, 2017. The JUTH HIV clinic, one of Nigeria’s oldest public HIV treatment centers, started enrolling patients for ART at the inception of Nigeria’s public ART program in 2002. The clinic received funding from the United States President’s Emergency Plan for AIDS Relief (PEPFAR) –23.2 or World Health Organization (WHO) HIV stage 4 disease or WHO HIV stage 3 disease with CD4 T-cell count < 350 cells/mm3. In 2010 adults with CD4 T-cell count < 350 cells/mm3, irrespective of WHO HIV stage or the presence of WHO HIV stage 3 or 4 HIV disease, became eligible for ART. In 2014, ART eligibility was extended to individuals with a CD4 T-cell count < 500 cells/mm3, HIV neuropathy, or coinfection with hepatitis B virus. By 2016, all patients with HIV, irrespective of CD4 T-cell count, were eligible for ART. The clinic monitored ART treatment response through scheduled CD4 T-cell count and HIV RNA measurement.The clinic offered free ART to all eligible patients during the study period. ART eligibility was determined by Nigerian ART treatment guidelines , 15, 24.Our analytic population comprised ART-naive patients with HIV 18 years or older, free of KS at or within 30 days of clinic enrollment .Our study was approved by the Institutional Review Board (IRB) of the Jos University Teaching Hospital Research and Ethics Committee and was ruled exempt by the IRB of Northwestern University, Chicago.We obtained de-identified, longitudinal data of patients who initiated HIV care from 2006 to 2016. The primary data source for this study was the JUTH HIV Clinic’s electronic health record system (EHRS). The EHRS was designed by the Harvard T.H-Chan School of Public Health using FileMaker Pro  and became operational in August 2004. The EHRS stored demographic, clinic, and laboratory records of all patients. To ensure data accuracy, the EHRS had built-in data error checks; additional checks were carried out by data managers each week.Our primary outcome was incident KS, which we defined as the first diagnosis of KS after 30 days of enrollment for HIV care. KS diagnosis was either clinical or histologic. We computed follow-up time from initiation of HIV care to KS diagnosis, last clinic visit, or December 31, 2017, whichever came first.The independent variable was the period of initiation of HIV care. Based on Nigeria’s ART eligibility expansion timeline , 15, 24,We computed the crude KS incidence by dividing the number of KS cases in each period by the person-time follow-up (years), multiplied by 1,000. We used a Poisson model with generalized estimating equations to calculate incidence rate ratios. The model assessed the occurrence of KS for a given year while accounting for person-year contributed by an individual by the end of a calendar year.We hypothesized that the expansion of ART eligibility led to a significant reduction in KS incidence. We used various Cox regression models to assess the hazard for incident KS between the pre-expansion (2006–2009) and post-expansion (2010–2016).We first ran bivariate analyses and then ran multivariable models adjusting for age, sex, and a marker of HIV severity . To adjust for the potential impact of ART on KS risk, we ran extended Cox models that adjusted for time-varying ART status and CD4 T-cell count or HIV viral load at baseline and at the time of ART initiation.We conducted subgroup analyses that included only patients who initiated ART during follow-up. We ran descriptive statistics and KS incidence in this sub-population. We also ran a Cox model from the time of initiation of HIV care to ART initiation (Pre-ART period) and another model that started at ART initiation to the censoring date (Post-ART period).N = 11,271). For the sensitivity analyses, we examined the effect of missing variables on the robustness of our analyses, using the multiple imputation method. Predictive mean matching was used to impute the missing data. Different analyses were done assuming missing at random or missing not at random. The details of the analyses are in the supplemental materials  received ART. The median time from initiation of HIV care to the commencement of ART was 38 (IQR 29–144) days. This time interval progressively reduced over time from 52 (IQR 42–231) days in 2006 to 14 (IQR 13–18) days by 2016  per 1000 person-years. Table p < 0.01).From 2006 to 2009, we identified 139 incident cases of KS during a follow-up period of 54,851 person-years, with a crude incidence of 2.53 per 1,000 person-years. From 2010 to 2016, 21 incident cases of KS were recorded over 13,271 person-years, with a crude incidence of 1.58 per 1,000 person-years. Based on the calendar year, KS incidence progressively declined over time , except for attenuation of the difference in the risk for KS in the time-varying models .We included data from 12,650 patients who received ART in this subgroup analysis. We report the characteristics of patients in Supplemental Table We identified 155 incident cases of KS over a follow-up period of 60,886 person-years, yielding an overall KS incidence of 1.02 (95% CI 0.79–1.31) per 1000 person-years. Time at risk was from initiation of HIV care. Although the incidence of KS was higher in patients who initiated HIV care in the pre-expansion compared to the post-expansion period (1.05 (95% CI 0.80–1.38) per 1000 person-years versus 0.89 (95% CI 0.49–1.61) cases per 1000 person-years) the difference was not statistically significant.n = 10,719). In the model which examined the risk for KS before ART initiation the risk for KS was 83% lower in patients who initiated HIV care in 2010–2016 compared to 2006–2009 . However, in analyses restricted to time on ART, the difference in KS risk between patients who initiated HIV care in 2010–2016 versus 2006–2009 narrowed .To elucidate the impact of ART use on KS risk, we conducted cox-regression analyses comparing the risk of KS in the pre-and post-expansion period before ART initiation and during ART use Table . We restAmong patients who enrolled for HIV care in a large HIV treatment center in Nigeria from 2006 to 2016, the incidence of KS declined during ART eligibility expansion. The crude incidence of KS declined from 2.53 to 1.58 per 1,000 person-years from 2006 - 2009 to 2010-16. After adjusting for age, sex, and CD4-Tcell count, the risk for KS was 52% lower among patients who initiated HIV care from 2010 to 2016 compared to 2006–2009. When we restricted our analyses to the period patients were on ART, although the adjusted incidence of KS remained lower in the post-expansion compared with the pre-expansion period, this difference was no longer statistically significant.Expanding access to ART has the potential to alter the epidemiology of HIV-associated malignancies in SSA. While several previous studies have evaluated the impact of ART on KS incidence , 25, theAdopting guidelines that stipulate earlier initiation of ART could contribute to a reduction in KS incidence through several mechanisms. One of the most important mechanisms may be preventing HIV-mediated CD4 T-cell depletion, which increases the risk for opportunistic infections and malignancies , 3, 18. Besides reducing the risk for KS through preventing CD4 T-cell depletion, early ART initiation also reduces HIV viral load. HIV viremia is hypothesized to contribute to increased KS risk by increasing the replication of HHV-8 and promoting angiogenesis and replication of spindle cells within the KS lesions –30. HoweTo better understand the impact of ART on KS risk, we restricted our analyses to time on ART. In these analyses, the risk for KS in the post-ART expansion era was 52% lower than in the pre–expansion after adjusting for age, sex, and CD4 T-cell count at the time of commencement of ART although this was not significantly significant. This result suggests that delay in ART initiation in the pre-expansion era, may have had led to residual increase in KS risk even among patients on ART. Delay in ART initiation has been associated with long term cancer risk . In addiOur findings have important implications for HIV-associated KS, particularly in SSA, where the incidence of HIV-associated KS remains high , 36, 37.This study has some limitations, which should be considered while interpreting our findings. First, the data analyzed were from a single institution where patients had access to specialist physician care and may not represent the level of care in other facilities. Because the data was not primarily collected for research, some variables were missing, which may have reduced the power of our study, particularly in our subgroup analyses. Another limitation is that we did not have histologic confirmation of KS, which may have led to the misclassification of some cases. Early, asymptomatic KS may have been missed, which may underestimate the burden of the disease. Also, we could not determine the prevalence of HHV-8 infection in our study population. Current HIV treatment guidelines do not recommend routine testing for HHV-8 infection, and commercial assays for its diagnosis are unavailable in Nigeria. Lastly, our study was in the pre-COVID-19 era and did not account for the effects of COVID-19 on HIV epidemiology.Despite these limitations, this study has several strengths. Our study is the largest on the incidence of HIV-associated KS in Nigeria, the country with the second-largest global HIV epidemic. Also, data for the analysis was obtained from an electronic health record system designed and maintained by the Harvard TH Chan School of Public Health, with mechanisms in place to ensure the high accuracy of data . We alsoIn conclusion, expanding ART eligibility was associated with a decline in the incidence of HIV-associated KS. Late presentation, however, remained pervasive. Since the emergence of COVID-19, studies have reported significant disruptions in the HIV care continuum, which may have more pronounced effects in low-resource settings. These disruptions could aggravate late presentation and reduce ART compliance among patients in care. A multifaceted approach aimed at early HIV diagnosis, linkage to care, timely ART initiation, and consistent ART supply is needed to successfully eliminate HIV-associated KS in settings with the twin epidemic of HIV and HHV-8.Additional file 1: Table S1. Characteristics of adults who initiated ART in Jos, Nigeria (2006-2018). Figure S1. Box plot of time from enrollment in care to initiation of antiretroviral therapy in adults with HIV in Jos, Nigeria (2006-2016). Table S2. Cox regression of predictors of Kaposi Sarcoma using Multiply Imputed Data from adults with HIV in Jos, Nigeria (2006-2016) . Table S3. Missing Data Pattern. Table S4. MICE model and corresponding populations and variables. Figure S2. MICE Mode A . Figure S3. MICE Model B  and MICE Model C . Figure S4. MICE Model D . Table S5. Cox Regression models of predictors of Kaposi Sarcoma among adults with HIV in Jos Nigeria (2006-2016). Table S6. Multivariate cox regression models of predictors of Kaposi Sarcoma among adults with HIV in Jos, Nigeria based on use of antiretroviral therapy (2006-2016)


Retraction Note: Cardiovascular Toxicology (2022) 22:916–928https://doi.org/10.1007/s12012-022-09768-0It appears a GADPH has been re-used in Figs. 1A, 1B and 2B.In Fig. 1, TNFa and NFkB look very similar to each other.In Fig. 2B, the first and last lanes of the GPx blot look very similar to each other.The Editor in Chief has retracted this article because of the following issues:The authors provided raw data which did not contradict these concerns and stated they did use the same GAPDH blot in the 3 figures. The Editor in Chief, therefore, has lost confidence in the integrity of the article's findings. None of the authors agree to this retraction.


COVID-19 is a worldwide pandemic caused by the highly infective SARS-CoV-2. There is a need for biomarkers not only for overall prognosis but also for predicting the response to treatments and thus for improvements in the clinical management of patients with COVID-19. Circulating cell-free DNA (cfDNA) has emerged as a promising biomarker in the assessment of various pathological conditions. The aim of this retrospective and observational pilot study was to investigate the range of cfDNA plasma concentrations in hospitalized COVID-19 patients during the first wave of SARS-CoV-2 infection, to relate them to established inflammatory parameters as a correlative biomarker for disease severity, and to compare them with plasma levels in a healthy control group.n = 21) during hospitalization in the University Medical Centre of Mainz, Germany between March and June 2020, and the cfDNA concentrations were determined by quantitative PCR yielding amplicons of long interspersed nuclear elements (LINE-1). The cfDNA levels were compared with those of an uninfected control group (n = 19).Lithium-Heparin plasma samples were obtained from COVID-19 patients (p < 0.001). Regarding clinical complications, the highest correlation was found between cfDNA levels and the myositis (p = 0.049). In addition, cfDNA levels correlated with the “WHO clinical progression scale”. D-Dimer and C-reactive protein (CRP) were the clinical laboratory parameters with the highest correlations with cfDNA levels.Plasma cfDNA levels in COVID-19 patients ranged from 247.5 to 6,346.25 ng/ml and the mean concentration was 1,831 ± 1,388 ng/ml (± standard deviation), which was significantly different from the levels of the uninfected control group (The results of this observational pilot study show a wide range in cfDNA plasma concentrations in patients with COVID-19 during the first wave of infection and confirm that cfDNA plasma concentrations serve as a predictive biomarker of disease severity in COVID-19. According to the World Health Organization (WHO), SARS-CoV-2 has caused more than 768.2 million cases worldwide to date  is the greatest worldwide pandemic of the 21 to date . Althoug to date , the ong to date . In addi to date . Disease to date . In addi to date , acute k to date  and gast to date . The WHO disease . There aThree years into the pandemic and despite the availability of effective vaccination the incidence of SARS-CoV-2-infection continues to show a wavy trend with potentially serious consequences in almost all age groups. Therefore, reliable biomarkers are still required. Biomarkers are not only needed for prognosis, but also for predicting response to treatment and thus for improving in the clinical management of patients with COVID-19. Recent studies have linked laboratory measures of hyperinflammation such as macrophage chemoattractant protein 1 (MCP-1), C-reactive protein (CRP) and interleukin-6 (IL-6), ferritin and procalcitonin (PCT) as strong predictors of disease severity in hospitalized patients with COVID-19 . Anotherhttps://www.medrxiv.org/content/10.1101/2021.04.29.21256291v1.Portions of this text were previously published as part of a preprint: ®; SAP, Weinheim, Germany, Nexus Swisslab, Berlin, Germany).A total of 21 patients hospitalized during the first wave of SARS-CoV-19 infection between March and June 2020 at the University Medical Centre, Mainz, Germany, were evaluated in this retrospective pilot study. This retrospective data collection is limited to the analysis of a highly inhomogeneous patient population due to patient consent for release or availability of their excess material, which also reflects the nature of a pilot study. When patients presented to the hospital with symptoms of COVID-19 disease, a nasopharyngeal swab was taken and tested for the presence of SARS-CoV-2 infection by polymerase chain reaction (PCR) in the in-house virology department. If the test was positive, the patients were included in the pilot study. Exclusion criteria were (1) age less than 18 years, (2) pregnancy, (3) breastfeeding women. Patients’ clinical data and laboratory results were reviewed retrospectively using the electronic hospital information systems (i.s.h.medn = 19) was included in the study in which SARS-CoV-2 infection was excluded by nasopharyngeal swab. The control group was subject to the exclusion criteria described above. To exclude other acute infections, CRP, leukocytes and platelets were also determined by laboratory tests. The cfDNA levels of the control group were compared with those of the COVID-19 cohort  Institute for Clinical Chemistry and Laboratory Medicine of the University Medical Centre, Mainz, Germany, as described  in accordance with the Declaration of Helsinki and by the local ethics committee of the “Landesärztekammer Rheinland-Pfalz” (reference numbers 2020-15116-retrospective). The Ethics Committee waived the requirement for informed consent for the retrospective cohort.2O , added to the reaction mix in a proportion of 1:7.5, and measured in technical replicates in a final volume of 5 µl. The final concentrations of the reaction mix were 0.04 IU Velocity Polymerase , 1.2 × Hifi Buffer , 0.15 × SYBR Green , 0.3 mM dNTPs , 140 nM of each primer. Samples were amplified using a Bio-Rad CFX384 system , using the following protocol: 98 °C for 2 min, followed by 35 cycles of 95 °C for 10 s (denaturation) and 64 °C for 10 s , followed by a melting curve from 70–95 °C with 0.5 °C increments for 10 s. No template control (NTC) samples, including H2O and mouse plasma, and two reference samples for calibration were included in each qPCR run. As described by p = 0.004). In lithium-heparin samples, no significant differences were observed between direct processing and extended storage (p = 0.5). The samples showed a concentration of 71.8 ± 33.1 and 80.2 ± 32.1 ng/ml (mean ± SD), respectively, demonstrating that lithium-heparin plasma samples are more suitable for cfDNA analysis than EDTA plasma samples.Lithium-heparin syringes  were used to collect blood plasma samples for routine laboratory analysis. Excess blood plasma was centrifuged at 3,746 × g for 10 min at room temperature in a safety level 2 laboratory (S2) within 2–3 h after collection. Plasma aliquots were stored at −80 °C prior to quantification of cfDNA concentrations. Plasma cfDNA concentrations were determined according to p-values are reported for the results. Spearman’s rank correlation coefficient was calculated analyzing the laboratory parameters in three different cohorts of cfDNA concentration . Spearman’s rho and p-values are given.Data analysis is descriptive. Means and standard deviations are presented for continuous variables, and absolute and relative frequencies and percentages are presented for categorical variables. A two-tailed Student’s T-test was performed to test the hypothesis that cfDNA levels in plasma samples are associated with clinical complications and risk factors in patients. Confidence intervals and To test the statistical correlation between the distribution of cfDNA and the classification of patients on the “WHO clinical progression scale”, cfDNA levels were log-transformed and a two-sided Spearman’s rank correlation test was performed. A two-tailed t-test was used to compare the mean values of cfDNA between the control group and each WHO group.2). The control group consisted of nine female and ten male subjects. The mean age was 43 ± 11 years and differed significantly between the two groups (p < 0.001). The control group had a mean BMI of 24.2 ± 4.3, again a significant difference between the groups (p = 0.009). There were no significant differences in other patient characteristics between the two groups.A total of 21 hospitalized patients with positive evidence of SARS-CoV-2 were evaluated in this pilot study, of whom twelve were male and nine were female. The patients were 68 ± 17 years old and on average overweight , renal (33%), pulmonary (38%) or immunological (14%) disease, arterial hypertension (57%), diabetes (19%) or adiposity (33%). In terms of pre-existing conditions, the control group had predominantly immunological pre-existing conditions (26.3%), mainly related to the thyroid. Overall, COVID-19 patients had significantly more pre-existing medical conditions than the control group. Patient characteristics are summarized in i.e., acute kidney injury (AKI). The inflammatory parameters CRP and procalcitonin (PCT) were markedly present in the majority of COVID-19 patients, reflecting hyperinflammation and/or secondary bacterial infection. Increased D-Dimer concentration was considered as an indicator of COVID-19 associated coagulopathy , followed by pulmonary complications including invasive ventilation and ARDS (43%), anemia and secondary infections . COVID-1p = 0.049). Interestingly, thromboembolism was found in patients with lower cfDNA concentrations and higher cfDNA concentrations were associated with absence of thromboembolism. Regarding patient outcome, cfDNA was significantly associated with in-hospital mortality (p < 0.001) (< 0.001) .The mean plasma cfDNA concentration in COVID-19 patients was 1,831 ± 1,388 ng/ml , with thp = 0.08, Associations between these three groups and complications were predominantly cardiovascular, renal, and pulmonary. Complications were most common in the hospitalized group with severe disease . The levThis retrospective observational pilot study compared plasma cfDNA levels between COVID-19 patients and an uninfected control group and explored correlations between cfDNA levels and clinical complications and outcomes as well as clinical blood laboratory measures in patients with COVID-19 during the first wave of infection. In our patient cohort, cfDNA levels were associated with patient outcome as measured by the WHO clinical progression scale, mortality, complications such as myositis, elevated D-dimer and CRP levels, thus reflecting outcome, organ complications and inflammation. Limitations of the present pilot study include the small cohort size with a broad spectrum of COVID-19 severity. This is mainly due to the unique human resource and structural conditions at the beginning of the COVID-19 pandemic. In such exceptional situations, the evaluation of small patient cohorts is extremely important in order to gain relevant clinical insights for future studies and therapy. We also analyzed a single plasma sample from each patient at individually different stages of their hospitalization. Therefore, it is not possible to generalize the results of this pilot study to all patients with COVID-19 and we cannot discuss on dynamic alterations of cfDNA data over time. Nevertheless, this pilot study has shown that even in a small sample, cfDNA levels correlate with disease severity as measured by the “WHO clinical progression scale”, clinical laboratory parameters and complications, and are significantly different from uninfected controls.Pre-existing conditions other than hypertension, obesity, and diabetes mellitus, which are considered typical risk factors for severe COVID-19 disease, were not assessed in this study. Although these pre-existing conditions were not correlated with cfDNA levels, we cannot exclude the possibility that other pre-existing conditions may have an overall effect on cfDNA levels that was not captured in this pilot study.cfDNA has been shown to be a valid marker for acute conditions such as stroke . Other pNotably, our assay amplifies repetitive LINE-1 elements, which comprise approximately 17% of the human genome. LINE-1 are retrotransposable elements that are epigenetically silenced in somatic cells and exhibit rare events of retrotransposition . LINE-1 Currently, there is no universal protocol for measuring cfDNA levels and presenting the data . This liIn addition, our results confirm previous studies showing that cfDNA is a liquid biopsy marker potentially suitable for predicting disease severity in patients with COVID-19 . AnotherDespite these limitations, our results are consistent with recently reported data on the predictive value of circulating  cfDNA for COVID-19 outcome . SignifiWe found the most significant correlations between cfDNA and CRP. Compared to CRP, cfDNA is a potentially more sensitive marker for monitoring and predicting disease progression. CRP is a non-specific acute-phase protein, produced by the liver in response to inflammation and tends to peak within 48 h of symptom onset. The half-life of CRP ranges from 12 to 24 h, depending on the underlying disease and factors such as age. On the other hand, cfDNA is released immediately after cell damage and may be useful in monitoring disease severity, progression, treatment response and predicting outcome in infectious diseases. The half-life of cfDNA in healthy individuals ranges from 15 min to 2 h, and can be increased in various health conditions, allowing for “real-time” analysis . FurtherAlthough both CRP and cfDNA are non-specific markers, cfDNA can provide information on the origin of cell damage through methylation analysis to specify organ damage.With our research showing that cfDNA correlates with disease severity in SARS-CoV-2 infection, further studies can be initiated to substantiate this advanced hypothesis.There are still hurdles to overcome before cfDNA can be used as a full-fledged biomarker for infectious diseases. Despite years of implementation in many medical fields, the determination of cfDNA is still very time-consuming compared to conventional inflammation parameters. The measurements are still not fully automated and require a high level of experience and expertise from laboratory professionals. In infectious disease monitoring, a combination of multiple biomarkers provides the best assessment of disease progression and response to treatment. CfDNA can add significant value to this portfolio. It should be noted that cfDNA is not only a biomarker for upstream pathophysiological mechanisms, but has also been proposed to trigger specific downstream effects. For example, cfDNA has been shown to be a regulator of the immune system , with diD-dimer is another biomarker for of coagulation and fibrinolysis  that is CfDNA levels in blood plasma samples from COVID-19 patients were significantly higher than those in an uninfected control group and correlated with the occurrence of clinical complications and disease severity as measured by the “WHO clinical progression scale”. Clinical laboratory measurements of D-dimer and CRP showed the highest correlations with cfDNA levels. The strong elevation of cfDNA in patients with COVID-19 in the first wave of disease confirms that cfDNA may be an important prognostic factor. This association has also been demonstrated in later waves of infection. However, inconsistent quantification methods make it difficult to compare measurements across studies. Whether cfDNA is a suitable method for monitoring therapy during the course of disease needs to be investigated in further studies. Therefore, prospective studies with quantification of cfDNA levels at defined time points during the course of disease are required.10.7717/peerj.16072/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.16072/supp-2Supplemental Information 2The patients’ laboratory parameters, comorbidities, duration of hospital- and ICU-stay and cfDNA measurements.Click here for additional data file.10.7717/peerj.16072/supp-3Supplemental Information 3Click here for additional data file.


The kinetics and adsorption isotherm models were used to investigate the removal of CV dye from aqueous solution using Zr-NDC. Langmuir adsorption isotherm model (R2 = 0.9880) describes the adsorption of CV dye onto Zr-NDC and the maximum equilibrium adsorption capacity (454.2 mg g−1) was achieved with the CV dye having an initial concentration of 100 mg L−1. The adsorption was found to follow pseudo-second-order kinetics (R2 = 0.9960) with a rate constant of 6.52 × 10−4 g mg−1 min−1. The effect of various parameters such as dye concentration, contact time, pH of dye solution, and MOF dose on the adsorption of dye was investigated. The study proved that the Zr-NDC is a promising adsorbent in the removal of CV dye from aqueous solution.Zirconium-2,6-naphthalenedicarboxylate metal-organic framework (Zr-NDC MOF) was prepared using ultrasound-assisted synthesis and tested for the adsorptive removal of crystal violet (CV) dye from aqueous solution. The pristine Zr-NDC was characterized using powder X-ray diffraction, scanning electron microscopy, thermogravimetric analysis, and energy-dispersive X-ray spectroscopy, Fourier-transform infrared, elemental analysis, and dynamic light scattering techniques. The maximum percentage removal of CV dye was found to be 99.45% with an initial dye concentration of 10 mg L Crystal violet (CV), also known as methyl violet 10B, is a synthetic basic cationic dye having antibacterial, antifungal, and anthelmintic properties. It is regarded as a biohazard substance and acts as a mitotic poison and a carcinogen. The presence of CV dye in water bodies results in undesirable colorations which, in turn, leads to low light penetrations for photosynthetic activities. Removal of CV from industrial wastewater is essential for the mitigation of environmental pollution and the safety of human and animal health .Several physicochemical methods such as adsorption, photochemical degradation, precipitation, electrochemical degradation, ion exchange, and biological methods were reported to effectively remove dyes from industrial wastewater –7. AmongMetal-organic frameworks (MOFs) are highly ordered porous crystalline materials. In recent years, zirconium-based MOFs have attracted the attention of the research community owing to their exceptionally high thermal, hydrothermal, mechanical, and chemical stability ,26. BaseCompared to various traditional and newly developed synthetic protocols, ultrasound-assisted synthesis for the preparation of MOFs is straightforward, convenient, and highly controllable . Synthes6O4(OH)4(NDC)6.xDMF by Maître group have been reported in the literature [Zr-NDC is one of the least studied members of the family of UiO-based MOF. It was synthesized using the solvothermal method by Zhang et al. in 2013 . So far,terature –34. Zr-Nterature –36. Therterature . So far,In the present study, Zr-NDC was synthesized for the first time using ultrasonication. Modulated synthesis strategy was used in the synthesis of MOF. The efficacy of pristine Zr-NDC for the effective removal of CV dye from the aqueous solution was investigated. The effect of various parameters such as dye concentration, adsorbent dose, contact time, and pH of dye solution on the adsorption was studied to optimize the application of Zr-NDC as an adsorbent in the removal of CV dye from aqueous solution. The adsorption isotherms and kinetics of adsorption in the adsorptive removal of CV dye by Zr-NDC were also studied.−1 range. Thermogravimetric analysis (TGA) measurement was carried out using Perkin Elmer STA 6000 thermal analyzer. Dynamic light scattering (DLS) measurements were performed on a Zetasizer  to determine the hydrodynamic parameters of the Zr-NDC MOF particles in water. Nitrogen adsorption-desorption isotherms were obtained on a BELSORP analyzer at −196 °C. A Shimadzu UV-2450 spectrophotometer was used to determine the absorbance of dye solutions in the visible region. An ultrasonic bath  was used for ultrasonication. Ultrasonic irradiation having a frequency of 40 kHz with a power of 150 watts was used. To alter the pH of the dye solution, 0.1 M HCl and 0.1 M NaOH solutions were used. A digital pH meter  was used to measure the pH of the solutions.All the chemicals were obtained from Sigma Aldrich and were used without any further purification. Powder X-ray diffraction (PXRD) patterns were recorded using a Bruker D8 Advance diffractometer equipped with monochromatized Cu Kα radiation (λ = 1.5406 Å) operated at 40 kV and 40 mA. Scanning electron microscopy (SEM) images were taken on a JEOL JSM-6390LA instrument. The acceleration voltage was set to 20 kV. Elemental analysis via energy-dispersive X-ray spectroscopy (EDS) was performed using an acceleration voltage of 10 kV so that zirconium (Lα = 2.042 keV), carbon (kα = 0.277 keV) and oxygen (kα = 0.525 keV) could be qualitatively determined. The elemental analysis for carbon, hydrogen, and nitrogen was performed with an Elementar Vario EL III analyzer. The Fourier transform infrared (FT-IR) spectra were obtained on Agilent Cary 660 instrument working in the transmission mode in the 400–4000 cm2.8H2O  in DMF (12 mL) and then acetic acid  was added. The resulting solution was placed in an ultrasonic bath and subjected to ultrasonic irradiation of 40 kHz (150 watts) for a period of 3 h. The temperature was monitored during the reaction and was observed to be less than 60 °C even after sonication for 3 h. After cooling down to room temperature, the as-synthesized white crystalline MOF was recovered by filtration. The solid material was washed three times with 10 mL of DMF followed by ethanol (3 × 10 mL) and acetone (3 × 10 mL). The material was dried overnight at room temperature and then activated by heating at 120 °C under a vacuum for 6 h. Under similar reaction conditions, crystalline Zr-NDC MOF was not obtained with the formic acid modulator. The attempts to obtain single crystals of MOF were also unsuccessful.The synthesis of Zr-NDC is outlined in Removal of CV dye from aqueous solution by adsorption was studied using as-synthesized Zr-NDC as the adsorbent. The adsorption studies were carried out using variable amounts of MOF, dye solutions having different initial concentrations, varying the pH of dye solution, and contact time of MOF with the dye solution. These studies were carried out at a temperature of 30 ± 2 °C. A standard solution of the dye was prepared by dissolving 1000 mg of the dye in 1000 mL of distilled water. A set of standard solutions was prepared using the stock solution. The absorbance maximum of dye solutions at 590 nm was measured and a calibration curve was plotted. In all the adsorption experiments, 100 mL of dye solution was used. The dye solution and MOF were mixed using a magnetic stirrer at 200 rpm. The adsorption efficiency,e (mg g−1), was calculated using qo is the initial dye concentration (mg L−1) and Ce is the dye concentration (mg L−1) at equilibrium. V is the volume of the dye solution (L) and M is the mass of Zr-NDC (g).where C−1. After the time interval of 12 h, the absorbance of each solution was measured and the Ce values were determined from the absorbance values using the calibration curve.Twenty milligrams of Zr-NDC was added to each of the 100 mL dye solutions having a concentration of 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 mg LThe percentage removal of dye was calculated using Adsorption isotherms were analyzed using three different adsorption isotherm models: Freundlich, Langmuir, and Temkin –39.Freundlich adsorption isotherm is expressed as F is the Freundlich constant and n is the constant whose value indicates how favorable the adsorption process is.Here KLangmuir isotherm is represented by m is the maximum adsorption capacity (mg/g) of the adsorbent (Zr-NDC) with the highest concentration of dye (100 mg L−1) used in the adsorption experiment and KL is the Langmuir adsorption constant.Here qTemkin isotherm is given by T is the adsorption heat constant (J) and KT is the equilibrium binding constant (L mg−1).Here B−1 and 20 mg of Zr-NDC as adsorbent was used. At the desired time (t), the dye solutions were withdrawn and the absorbance was measured. The dye amounts adsorbed at time t, qt (mg g−1) was calculated using For the kinetic study, 100 mL of dye solution having a concentration of 10 mg Lt is the dye concentration (mg L−1) at time t (min).Here CThree kinetic models, pseudo-first-order, pseudo-second-order, and intraparticle diffusion were applied to study the rate of removal of dye in the adsorption process . The thrPseudo-first-order kinetics,Pseudo-second-order kinetics,Intraparticle diffusion kinetics,1 (min−1), k2 (g mg−1 min−1) and ki (mg g−1 min−1/2) are the rate constants for pseudo-first-order, pseudo-second-order, and intraparticle diffusion respectively. C is the intraparticle diffusion constant.Here kThe pristine Zr-NDC was characterized using different characterization techniques. The MOF was highly crystalline and the crystals had spherical morphology with homogenous size distribution as evident from PXRD patterns and SEM images in −1 was compared with that of 2,6-naphthalenedicarboxylic acid (free organic linker) in −1) was shifted to a lower wavenumber in the MOF spectrum, and two strong bands observed at 1406 and 1600 cm−1 can be attributed to symmetric and asymmetric stretching vibrations of carboxylate ions which were due to the reaction between organic linkers and the metal ions. The absorption bands in the region 2500–3000 cm−1 corresponding to the stretching vibrations of the O-H bonds in the free organic linker were no longer present in the MOF spectrum. The absorption band at 556 cm−1 and 453 cm−1 were ascribed to the asymmetric stretching vibration of Zr-(OC) and bending vibration of (OH)-Zr-(OH) bonds respectively. Based on the previous reports, the C=O stretching frequency observed at 1654 cm−1 corresponds to DMF. Since, in liquid DMF, this characteristic band was located around 1675 cm−1, the shifting of the absorption band to a lower wavenumber suggests that the DMF molecules were coordinated to the metal nodes of the Zr-NDC through the carbonyl groups [The FT-IR spectrum of Zr-NDC recorded in the region 400–4000 cml groups ,43.2 is formed as the final solid product due to the decomposition of the organic linker and acetic acid from the framework. The characteristic decomposition pattern observed in the TGA curve supports the composition of the synthesized Zr-NDC. From TGA analysis, it was evident that the pristine Zr-NDC was defective and there were missing organic linkers in the MOF structure. Acetic acid and DMF are coordinated to the metal as compensating ligands to compensate for the loss of coordination due to the missing organic linkers. FT-IR spectroscopy and elemental analysis data also support these facts.6O4(OH)4(NDC)4(CH3COO)(C2H5OH)3 (DMF)2. Calculated, %: C, 39.68; H, 3.36; N, 1.49.Elemental analysis: Found, %: C, 38.13; H, 2.10; N, 1.42. Zr2/g, 0.47 cm3/g, and 8.29 nm respectively.To explore the solution-based applications of MOF particles such as drug delivery, Zr-NDC particles were characterized in solution with DLS . The par−1. The equilibrium time for adsorption was 12 h. The effect of the initial concentration of dye on the adsorption by Zr-NDC was shown in −1 to 100 mg L−1. As reported in the earlier study, the decrease in the percentage removal of dye can be attributed to the saturation of adsorption sites with the initial dye concentration of 10 mg L−1 [Using 20 mg of Zr-NDC adsorbent, the adsorption experiment was carried out by varying the initial concentration of CV dye in the range of 10–100 mg L0 mg L−1 .−1 CV dye solution. The percentage removal of dye was increased from 39.21% to 99.45% with the increase in the dose of Zr-NDC from 5 mg to 20 mg due to the increase in surface area of the MOF. Maximum dye removal percentage was achieved with 20 mg of MOF dose.The effect of the Zr-NDC dose on the percentage removal of CV dye in the adsorption experiments was shown in The variation in the percentage of dye removal with the contact time was shown in −1 and Zr-NDC adsorbent dose of 20 mg at a temperature of 30 ± 2 °C for 12-h equilibrium time. The variation in percentage removal of dye by adsorbent with the change in the pH of the solution is shown in pH is one of the key factors affecting the efficiency of adsorbents in the removal of dye from the aqueous solution. Earlier studies revealed that variation in pH of the solution will alter the surface properties of the adsorbent and the degree of ionization of the absorptive molecules . To stud2 = 0.9880) was the best fit compared to Freundlich (R2 = 0.9525) and Temkin (R2 = 0.9034) models. Hence, the Langmuir adsorption of CV dye from aqueous solution onto Zr-NDC can be best described by the Langmuir adsorption isotherm model. From the slope of the plot in m, cal) of the Zr-NDC adsorbent was found to be 458.71 mg g−1. This value was very close to the experimental maximum adsorption capacity  value of 454.20 mg g−1. From the intercept of the plot, KL was found to be 1.172.The three most widely applied isotherm models, Freundlich, Langmuir, and Temkin, were applied to find out the adsorption performance of Zr-NDC. Adsorption isotherm models were used to provide information about the mechanism of adsorption, evaluation of the performance of the adsorption process, and capacity of Zr-NDC as an adsorbent. These three adsorption isotherms were represented by Figures 4L) was calculated using An important characteristic of the Langmuir adsorption isotherm, separation factor  and KL is the Langmuir constant.Here C−1) used in the adsorption experiment, the calculated value of RL was 0.00846. Since the RL obtained was between 0 and 1 (0 < 0.00846 < 1), the adsorption process was favorable [With the highest concentration of dye (100 mg Lavorable . The resThe adsorption capacity of Zr-NDC for the adsorption of CV dye was compared with various other MOF adsorbents reported in the literature as shown in 2), it was found that the pseudo-second-order kinetic model is the best fit for the adsorption kinetics data obtained in the present study. The value of R2 (0.9960) for the pseudo-second-order kinetic plot is higher than the R2 values of linear plots of the other two kinetics models. The calculated maximum adsorption capacity  was in good agreement with the experimental value . The pseudo-second-order rate constant was determined to be 6.52 × 10−4 g mg−1 min−1. The results obtained in the kinetics studies suggest that the adsorption of CV from aqueous solution onto the Zr-NDC surface proceeds via chemisorption [To study the adsorption kinetics, three kinetic models, pseudo-first-order, pseudo-second-order, and intraparticle diffusion were used. These three models were represented by Figures 7sorption .Based on the analyses of adsorption isotherm data, it is clear that the Zr-NDC has a good adsorption capacity for CV. Even though a detailed study is necessary to understand the adsorption mechanism, based on the earlier studies, the adsorption may be primarily due to the electrostatic interaction between anionic Zr-NDC and the cationic CV . CV is a−1) was achieved with the CV dye having an initial concentration of 100 mg L−1. Zr-NDC was found to be a highly efficient adsorbent for the removal of CV dye from aqueous solution.This study reports the successful synthesis of Zr-NDC for the first time using environmentally friendly ultrasonic-assisted synthesis. The reaction conditions were optimized for the synthesis of Zr-NDC via modulated synthesis approach using 30 equivalents of acetic acid. The removal of CV dye from aqueous solution using pristine Zr-NDC was investigated. A study of adsorption kinetics and adsorption isotherm models revealed that pseudo-second-order kinetics and Langmuir adsorption isotherm model best describe the adsorption of CV dye onto Zr-NDC. The maximum equilibrium adsorption capacity (454.2 mg g


The graft copolymer self-assembles in aqueous media, forming a three-dimensional (3D) network through polyelectrolyte complexation of the oppositely charged PAA and PLL segments. Rheological investigations showed that the hydrogel exhibits interesting properties, namely, relatively low critical gel concentration, elastic response with slow dynamics, remarkable extended critical strain to flow, shear responsiveness, injectability, 3D printability and self-healing. Due to the weak nature of the involved polyelectrolyte segments, the hydrogel properties display pH-dependency, and they are affected by the presence of salt. Especially upon varying pH, the PLL secondary structure changes from random coil to α-helix, affecting the crosslinking structural mode and, in turn, the overall network structure as reflected in the rheological properties. Thanks to the biocompatibility of the copolymer constituents and the biodegradability of PLL, the designed gelator seems to exhibit potential for bioapplications. We report the rheological behavior of aqueous solutions of a graft copolymer polyampholyte, constituted of polyacrylic acid (PAA) backbone grafted by Poly(L-lysine) (PAA- The macromolecular design has been established as a molecular tool to create three-dimensional networks formed by the self-organization of polymeric materials through various reversible interactions, forming weak non-covalent bonds. These networks swollen in aqueous media form soft materials, the so-called hydrogels, that have attracted immense attention due to a large variety of potential applications ,2,3,4,5.The charge-driven assembly concept has been used to design networks based on interactions between oppositely charged moieties, the bond energy of which ranges from 5 to 200 KJ/mol . Synthet2), exhibiting thus the pH-dependent degree of ionization, are very interesting candidates as charged segments for designing macromolecules capable of forming networks through ionic complexation /[COO−] molar ratio decreases with increasing pH and tends to zero at pH 12. This is reflected in the variation of the z-potential of the solutions presented in 3+]/[COO−] molar ratio decreases. Thus, in the pH range investigated, the formed network is always of a negatively charged polyelectrolyte nature.The variation of pH significantly affects the degree of ionization of the graft copolymer constituents and, in turn, their conformation. Moreover, it influences the strength of ionic interactions between the oppositely charged blocks (PAA-PLL attractive) forming the polyelectrolyte complexes and between similarly charged segments (PAA-PAA repulsive) as well. Specifically, by increasing pH, the PAA backbone ionization degree increases by deprotonation of the carboxylic groups, imposing a conformational transition from random coil to extended rod-like chain conformation . On the α-helix upon varying pH [It is well known that the PLL polypeptide is subjected to secondary chain conformational transition from random coil to rying pH ,42. Therα-helix) reveal a conformation transition from random coil to α-helix, contrary to what is observed in pure PLL, exhibiting random coil in this range of pH. However, at pH 7.4, the broadening of the band at 208 nm towards 204 nm should be attributed to the coexistence of some random coil conformations [α-helix transition occurs at a pH higher than 10.8 (pKa of PLL), where the polypeptide becomes uncharged. Indeed, this is valid at pH 11.8, as depicted in α-helix conformation of the PLL side chains at the intermediate pH should be attributed to the ionic interactions with the negatively charged carboxyl groups of PAA segments that neutralize the positively charged amine groups of PLL stabilizing hence the helical structure. This is in good agreement with the results of Shinoda et al. exploring the complexation between PAA and PLL homopolymers [α-helix of PLL in the complex, which is reasonable to occur also in our case. Therefore, the main pH effect in the structure of PAA-g-PLL networks is the conformational transition of the PLL secondary structure, which affects the association mode of the crosslinks of the network. That is, PAA random coil—PLL random coil (pH 5.5), PAA left-handed superhelix—right-handed a-helix of PLL  and PLL α-helix hydrophobic association (pH 11.8) (α-helixes.The CD spectrum at pH 5.5 shows the characteristic pattern of a random coil (single strong negative band at 200 nm) in accordance with the spectrum of pure PLL . At pH 7rmations . The ranpolymers . In the pH 11.8) c. These pH 11.8) c. For inporeD) . As it is known, the elastic modulus is inversely proportional to the mesh size ξ of the network, G = PKT/ξ (where P is the number of overlapping network strands inside the correlation volume [ξ implies a denser network which seems to lead to a higher poreD (see schematics in Scanning electron microscopy (SEM) was performed in two different freeze-dried hydrogels, prepared at pH 7.4 and 5.5 (Cp = 5 wt%), relevant to potential bioapplications. In Dpore) . Dpore in volume , due to NaCl up to 0.45 M. The high-frequency G′ increased upon adding 0.15 M NaCl and then decreased steadily upon further increasing CNaCl. The initial overshooting of G′ with CNaCl is not rare, as has been observed in other network systems formed by charge-driven assembly [NaCl strengthens the electrostatic screening, increasing hence the chain flexibility of the PAA segments, which seems to impose intermolecular to intramolecular transitions, decreasing G′ eventually.Polyelectrolytes and their ionic complexations are sensitive to the ionic strength of the solution. We thus proceeded to explore the influence of the presence of salt on the rheological properties of the hydrogels, prepared at fixed pH 7.4. In all cases, elastic hydrogels were observed as the G′ predominates the G″ in the entire frequency range investigated. In assembly ,34. At l2)4 sequences in their pendant groups. This is in qualitative agreement with the results obtained by the co-assembly of PGA-PEG-PGA (PGA is poly(L-glutamic acid) and PLL-PEG-PLL [The same behavior was observed in the complex viscosity, which is affected by the exchange dynamics of PLL from the crosslinks (complexes with PAA) besides the number density of crosslinks. The frequency sweep data do not show the appearance of G′/G″ crossover at low frequencies suggesting that the relaxation of the network is still very slow. In other words, the terminal relaxation time, which is mainly correlated with the lifetime of the crosslinks, is much higher than the time of observation. We speculate that the reduction in the attractive ionic interactions among the oppositely charged segments with increasing ionic strength is compensated by the hydrophobic ones to some extent due to the L-lysine hydrophobic nature, bearing -(CH-PEG-PLL .c above which the formulation flows (G″ > G′). As illustrated in c increased remarkably with CNaCl. Especially by adding 0.45 M NaCl in the aqueous medium, γc increased more than two-fold. This might be ascribed to the increase in the chain flexibility of the elastically active PAA segments because of the electrostatic screening of the repulsive interactions along the chain, allowing higher extensibility. Moreover, the weakening of the ionic interactions among the PAA/PLL segments allows easier rearrangements of the crosslinks during the strain evolution.Another interesting consequence of the effect of increasing ionic strength is the increase in the critical strain γc). In the third step, by applying low strain again within the LVR, the hydrogel was recovered instantly. A short delay can be observed in the evolution of G′, which is recovered about 90% after 60 s. Finally, in the fourth step, by applying the same strain as the second step, the gel flows again, behaving similarly, which implies very good responsiveness to shear. All the data in To explore the self-healing ability of hydrogel, a two-step consecutive experiment was conducted. In the first step, the sample was subjected to a strain sweep beyond the critical strain (at γ = 4000%) to disrupt the 3D structure allowing flow, followed by a second step, setting γ at 5%, within the LVR. As observed in −1 followed by a third step at physiological temperature (37 °C) applying a shear rate of 0.01 s−1. The first low-value approach to the zero-shear viscosity, simulates gel at rest, and the second value exemplifies the shear rate applied through a 28-gauge syringe needle [−1 at 25 °C and recovers to the previous values when the shear rate is reverted to 0.01 s−1 at 37 °C, simulating conditions after injection at physiological temperature. More importantly, the viscosity changes instantly upon varying the shear rate, implying excellent shear responsiveness of the hydrogel in accordance with the data of the oscillatory measurements  and under shear rates of 0.01 and 17.25 se needle . Figure urements . The steurements .P implying that it is a factor that could be used to tune injectability. For instance, at CP = 3 wt%, the viscosity under a shear rate of 17.25 s−1 (injection conditions) drops to the order of 1 Pa·s which seems to meet the requirements for injectability. Nevertheless, the shear-induced viscosity decrease demonstrated in The same behavior was observed for all the formulations of different gelator concentrations, as presented in g-PLL-based hydrogel is its adhesive capability, which was optically observed on the cone and plate rheometer. As illustrated in the successive images of c  precursor form hydrogels in aqueous media. These two gelators have the same macromolecular topology, differing only in the nature of the PLL functional repeating units, i.e., NH2 (ionogenic) vs. -NH-boc (hydrophobic). Thus, it would be useful to compare their hydrogel properties, provided that they self-assemble in water through different interactions, i.e., ionic vs. hydrophobic. The PAA-g-P(boc-LL) hydrogel structure is constituted of densely packed swollen hydrophobically crosslinked finite networks (microgels), while the PAA-g-PLL copolymers form a 3D network through ionic complexation. This significant structural difference produces remarkable differences in their rheological properties, namely lower critical gel concentration and critical strain to flow (γc) (about three orders of magnitude) for the PAA-g-P(boc-LL), but remarkably higher moduli at comparable polymer concentrations.The PAA-gel, determined at CP = 2.35 wt%. Dynamic and steady-state shear rheology were used to explore the hydrogel properties. The hydrogels exhibit a nearly elastic response with slow dynamics and remarkable extended critical strain, higher than 1000%, above which the hydrogels flow. Thanks to the weak polyelectrolyte nature of the graft copolymer constituents, the hydrogel exhibits properties sensitive to pH. More importantly, provided that the PLL polypeptide side chains adopt secondary structures affected by the ionic interactions with PAA and/or the medium pH, different network structures were observed. Particularly, upon increasing pH, the association mode of the crosslinks changes from PAA random coil—PLL random coil (pH 5.5) to PAA left-handed superhelix—PLL right-handed a-helix  and finally to PLL α-helix association (pH 11.8). In the hydrogel features, we should mention the resistance to heat, the sensitivity to ionic strength and the excellent responsiveness to shear. The latter property provides injectability and 3D printability. Due to physical crosslinks of ionic complexation that can be disrupted under shear deformation and be reformed rapidly at rest, the hydrogel also exhibits self-healing properties. Thanks to the biocompatibility of the copolymer constituents and the biodegradability of PLL, the designed gelator could be considered a good candidate for potential bioapplications.A polyampholyte graft copolymer, constituted of polyacrylic acid (PAA) backbone grafted by PLL, bearing negatively and positively ionogenic moieties, respectively, was designed and evaluated as a gelator in aqueous media. This segmented graft-type polyampholyte self-assembles spontaneously in water mainly through ionic interactions among the oppositely charged segments, forming a 3D network at concentrations above a percolation threshold Cg-P(boc-LL) graft copolymer (bearing boc-protected -NH2) was synthesized by the grafting onto method using carbodiimide chemistry. Details about this process are reported elsewhere [w = 575,202 Da and grafting density 8% mol . Trifluoroacetic acid (TFA), Hydrochloric acid , Sodium Hydroxide , deuterated solvent Deuterium Oxide (D2O)  were purchased from Sigma-Aldrich  and used without further purification. Ultra-pure three-distilled water (3D-H2O) was obtained by means of an ELGA Medica R7/15 device.PAA-lsewhere . The gra−3 mol) PAA-g-P(boc-LL) were weighted and dissolved in 250 mL of 3D-H2O and left under stirring for 3 days at 25 °C. A homogenous gel was formed. Afterward, 40 mL of TFA (0.414 mol/in excess) was added and immediately, the gel was transformed into a clear solution. The deprotection reaction was left to proceed for 3 h in a cooling bath due to its exothermic character. The resulting solution was dried by using a Rotary Evaporator to remove the remaining amounts of TFA, and then 200 mL of 3D water was added to dissolve the polymer. To isolate the graft copolymer PAA-g-PLL from by-products , a dialysis procedure in neutral pH against ultra-pure water using a dialysis membrane  was held for three days. The final PAA-g-PLL product was freeze-dried and collected from a vacuum drying oven.Simple carbamate hydrolysis in acidic conditions occurred for the removal of boc species from protected amines in the side polypeptide chains. Initially, 0.7518 g (1.30 × 10g-PLL in terms of grafting density was accomplished by Proton Nuclear Magnetic Resonance Spectroscopy (1H-NMR), using a BRUKER AVANCE III HD PRODIGY ASCEND TM 600 MHz spectrometer  at 25 °C.The check of the removal of Boc residues and the characterization of the graft copolymer PAA-g-PLL graft copolymer was dissolved in deuterated water (D2O), and the characteristic 1H-NMR spectra are presented in An amount of PAA-g-PLL graft copolymer were prepared at different concentrations, various pH conditions and salt concentrations, as shown in 2HPO4/KH2PO4 (PB) 1 mM and pH = 7.4, and the pH value was controlled by adding HCl or NaOH solution (1M). For examination of the salt effect, the graft copolymer solutions with specific ionic strength were formed by adjusting the NaCl concentration in solute (PB).Aqueous solutions of PAA-The rheological studies of the graft copolymer hydrogels and solutions were conducted by using a stress-controlled AR-2000ex  rheometer, with a cone and plate geometry , equipped with a Peltier plate system that allows the precise control of temperature (±0.1 °C) and a solvent trap that prevents changes in concentrations due to water evaporation. The experiments were performed at 25 °C.p = 5% wt) at pH = 5.5 and pH = 7.4 were prepared in the same manner above mentioned in Hydrogel samples . A 0.1 cm cell well was filled with polymer aqueous solutions of different pH (controlled by adding 1 M HCl or NaOH)and at concentrations close to 1.0 × 10Z-potential measurements of aqueous polymer solutions of different pH were conducted by using a Brookhaven Instruments Nanobrook Omni  system operating at λ = 640 nm and with 40 mW power, with the Smoluchowski method, operating in Electrophoretic Light Scattering (ELS) mode.